There are many different types of assays that employ nucleic acid hybridization probes and that utilize for signal generation a change in the fluorescence of a fluorophore due to a change in its interaction with another molecule or moiety brought about by changing the distance between the fluorophore and the interacting molecule or moiety.
These assays rely for signal generation on fluorescence resonance energy transfer, or "FRET", according to which a change in fluorescence is caused by a change in the distance separating a first fluorophore from an interacting resonance energy acceptor, either another fluorophore or a quencher. Combinations of a fluorophore and an interacting molecule or moiety, including quenching molecules or moieties, are known as "FRET pairs." The mechanism of FRET-pair interaction requires that the absorption spectrum of one member of the pair overlaps the emission spectrum of the other member, the first fluorophore. If the interacting molecule or moiety is a quencher, its absorption spectrum must overlap the emission spectrum of the fluorophore. Stryer, L., "Fluorescence Energy Transfer as a Spectroscopic Ruler," Ann. Rev. Biochem. 1978, 47: 819-846 ("Stryer, L. 1978"); BIOPHYSICAL CHEMISTRY part II, Techniques for the Study of Biological Structure and Function, C. R. Cantor and P. R. Schimmel, pages 448-455 (W. H. Freeman and Co., San Francisco, U.S.A., 1980) ("Cantor and Schimmel 1980"), and Selvin, P. R., "Fluorescence Resonance Energy Transfer," Methods in Enzymology 246: 300-335 (1995) ("Selvin, P. R. 1995"). Efficient, or a substantial degree of, FRET interaction requires that the absorption and emission spectra of the pair have a large degree of overlap. The efficiency of FRET interaction is linearly proportional to that overlap. Haugland, R. P., Yguerabide, Jr., and Stryer, L., "Dependence of the Kinetics of Singlet-Singlet Energy Transfer on Spectral Overlap," P.N.A.S. (U.S.A.) 63: 24-30 (1969) ("Haugland et al. 1969"). The cited art teaches that to obtain a large magnitude of signal, a high degree of overlap is required. FRET pairs, including fluorophore-quencher pairs, have been chosen on that basis.
One suitable FRET pair disclosed in Matayoshi et al. 1990, Science 247: 954-958, includes DABCYL as a quenching moiety (or quenching label) and EDANS as a fluorophore (or fluorescent label). The absorption spectrum of DABCYL has a high degree of overlap with the emission spectrum of EDANS, making these two a good FRET pair. Despite the recognized advantage of using a FRET pair that includes a quencher that does not itself fluoresce, such as DABCYL, very few such FRET pairs have been identified. In general, the number of fluorophore-quencher pairs is extremely limited because of the need for a high degree of spectral overlap. Additional such pairs would be highly desirable for a number of reasons, including flexibility in assay design and distinguishing signals in multiplex assays.
A variety of labeled nucleic acid hybridization probes and detection assays that utilize FRET and FRET pairs are known. One such scheme is described by Cardullo et al. (1988), P.N.A.S. 85: 8790-8794 and in Heller et al. EP 0070685. A2, claiming priority of U.S. 284469 filed on Jul. 17, 1981. It uses a probe comprising a pair of oligodeoxynucleotides complementary to contiguous regions of a target DNA strand. One probe molecule contains a fluorescent label, a fluorophore, on its 5' end, and the other probe molecule contains a different fluorescent label, also a fluorophore, on its 3' end. When the probe is hybridized to the target sequence, the two labels are brought very close to each other. When the sample is stimulated by light of an appropriate frequency, fluorescence resonance energy transfer from one label to the other occurs. FRET produces a measurable change in spectral response from the labels, signaling the presence of targets. One label could be a "quencher," which in this application is meant an interactive moiety (or molecule) that releases the accepted energy as heat.
Another solution-phase scheme utilizes a probe comprising a pair of oligodeoxynucleotides and a FRET pair. However, here the two probe molecules are completely complementary both to each other and to complementary strands of a target DNA (Morrison and Stols, "Sensitive Fluorescence-Based Thermodynamic and Kinetic Measurements of DNA Hybridization in Solution,"Biochemistry 32: 309-3104 (1993) and Morrison EP 0 232 967 A2, claiming priority of U.S. application Ser. No. 817,841, filed Jan. 10, 1986. Each probe molecule includes a fluorophore conjugated to its 3' end and a quenching moiety conjugated to its 5' end. When the two oligonucleotide probe molecules are annealed to each other, the fluorophore of each is held in close proximity to the quenching moiety of the other. With the probe in this conformation, if the fluorophore is then stimulated by light of an appropriate wavelength, the fluorescence is quenched by the quenching moiety. However, when either probe molecule is bound to a target, the quenching effect of the complementary probe molecule is absent. In this conformation a signal is generated. The probe molecules are too long to self-quench by FRET when in the target-bound conformation.
A solution-phase scheme that utilizes FRET pairs and the phenomenon known as strand displacement is described by Diamond et al. U.S. Pat. No. 4,766,062; Collins et al. U.S. Pat. No. 4,752,566; Fritsch et al. U.S. Pat. Nos. 4,725,536 and 4,725,537. Typically, these assays involve a probe comprising a bimolecular nucleic acid complex. A shorter single strand comprising a subset of the target sequence is annealed to a longer single strand which comprises the entire target binding region of the probe. The probe in this configuration thus comprises both single-stranded and double-stranded portions. Diamond et al. proposed that these probes may further comprise either a .sup.32 P label attached to the shorter strand or a fluorophore and a quencher moiety which could be held in proximity to each other when the probe conformation is that complex.
Another type of molecular probe assay utilizing a FRET pair is described in European Patent Application 0 601 889 A3, publication date Jun. 15, 1994, which claims priority of Bagwell U.S. patent application Ser. No. 990,298, filed Dec. 10, 1992.
Another type of nucleic acid hybridization probe assay utilizing a FRET pair is the so-called "TaqMan" assay described in Gelfand et al. U.S. Pat. No. 5,210,015, and Livak et al. U.S. Pat. No. 5,538,848. The probe is a single-stranded oligonucleotide labeled with a FRET pair. In a "TaqMan" assay, a DNA polymerase releases single or multiple nucleotides by cleavage of the oligonucleotide probe when it is hybridized to a target strand. That release provides a way to separate the quencher label and the fluorophore label of the FRET pair. According to Livak et al. "straightening" of an end-labelled "TaqMan" probe also reduces quenching.
Yet another type of nucleic acid hybridization probe assay utilizing FRET pairs is described in Tyagi et al. copending United States patent application Ser. No. 08/439,819 now U.S. Pat. No. 5,925,517 (and counterpart applications outside the United States, including PCT Application No. WO 95/13399), which utilizes labeled oligonucleotide probes, which we have come to refer to as "Molecular Beacons." Tyagi, S. and Kramer, F. R., "Molecular Beacons: Probes that Fluoresce upon Hybridization," Nature Biotechnology 14: 303-308 (1996). A Molecular beacon probe is an oligonucleotide whose end regions hybridize with one another in the absence of target but are separated if the central portion of the probe hybridizes to its target sequence. The rigidity of the probe-target hybrid precludes the simultaneous existence of both the probe-target hybrid and the intramolecular hybrid formed by the end regions. Consequently, the probe undergoes a conformational change in which the smaller hybrid formed by the end regions disassociates, and the end regions are separated from each other by the rigid probe-target hybrid.
Aspects of this invention include probes containing non-FRET fluorophore-quencher pairs and chromophore pairs useful in assays; improved assays, including multiplexed assays, utilizing such pairs of molecules or moieties; and assay kits that include such pairs.